Summary: Rabbit Preimmune Repertoire Development We study genes of the rabbit immune system using techniques of molecular biology and immunology (1). In species such as mouse and human, generation of combinatorial diversity through use of different VH and VL genes in immunoglobulin VHDJH and VLJL rearrangements can be a major contributor to the primary antibody repertoire. In rabbits, the contribution of the combinatorial mechanism to heavy chain diversity is minimal as only a few VH genes are rearranged and expressed. This resembles chicken antibody formation. Rabbit appendix and chicken bursa of Fabricius are primary lymphoid organs where the B cell antibody repertoire develops in germinal centers mainly by a gene conversion-like process. As in the chicken, the 3-prime most VH1 gene is rearranged in most rabbit B lymphocytes. Somatic hypermutation and gene conversion contribute to primary diversification in appendix of young rabbits or in bursa of Fabricius of embryonic and young chickens and also to secondary diversification during immune responses in germinal centers (GCs)(2). We previously showed that diversification patterns in the clones from appendix were strikingly different from those found in splenic GCs where an immunizing antigen was driving the expansion and selection process toward high affinity. Clonally related appendix B cells developed different amino acid sequences in each complementarity-determining region (CDR) including CDR3 whereas dominant clones from spleen underwent few changes in CDR3. The variety of combining sites generated by diversification within individual appendix clones suggests that at least some clonal expansion and selection, known to require normal gut flora, may be driven through indirect effects of microbial components rather than solely by their recognition as specific foreign antigens. In collaboration with Dr. Ramit Mehr, we used a novel method for analyzing lineage tree shapes, using terms from graph theory to study diversification in rabbits and chickens. When lineage tree shapes were analyzed to quantify the differences between primary and secondary diversification in rabbits, the analyses indicated that primary diversification appears to occur at a constant rate in the appendix and the type of antigen-specific selection seen in splenic GCs is absent. This supports the view that a primary repertoire is being generated within the expanding clonally related B cells in appendix of young rabbits and emphasizes the important role that gut associated lymphoid tissues may play in early development of mammalian immune repertoires. The data also indicate a higher rate of hypermutation in rabbit during immune responses in splenic GCs, such that the balance between hypermutation and selection tends more towards mutation and less towards selection in rabbit compared to murine GCs (3). Rabbit Immune Repertoires for Generation and Humanization of Therapeutic Monoclonal Antibodies The rabbit immune repertoire has long been a rich source of diagnostic polyclonal antibodies. Now it also holds great promise as a source of therapeutic monoclonal antibodies. Rabbits with the rare b9 and bas allotypes are excellent sources for therapeutic monoclonal antibodies. We generated rabbit polyclonal antibodies and from these rabbits, the laboratory of Dr. C. Rader isolated monoclonal antibodies selective for all three members of the Nogo receptor (NgR) family. NgR family members are cell surface proteins involved in the development, plasticity, and regeneration of the central nervous system. The use of our antibodies to characterize NgR2 was recently published (4). Vascular endothelial growth factor (VEGF) and its receptors have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. Murine tumor angiogenesis models and receptor-specific antibodies are required to facilitate evaluation of the roles of VEGF receptors in mouse models of human cancer. We developed rabbit antibodies that cross-react with mouse (Flk-1) and human (KDR) VEGFR2. High-affinity, species cross-reactive, VEGFR2-specific Fabs were selected from an antibody phage display library generated from an immunized b9 allotype rabbit. The selected chimeric rabbit/human Fabs were found to bind to KDR and Flk-1 with nanomolar affinity. Three selected Fabs detected KDR expression on human endothelial cells as well as Flk-1 on murine endothelial cells. The availability of anti-VEGFR2 Fab with species cross-reactivity will help to decipher functional role of KDR/Flk-1 in tumor biology as well as facilitate the preclinical evaluation of the suitability of KDR/Flk-1 for drug targeting. This report further underscores our earlier finding that b9 allotype rabbits are excellent sources for generating high affinity cross-reactive antibodies with therapeutic potential (5).